Our overall goal in this project is to make available inexpensive neutralizing antibodies against Botulinum neurotoxin in large quantity. We will combine our experience in producing functional antibodies in transgenic plants with three neutralizing antibodies isolated by our collaborator, Dr. James D. Marks of the UC San Francisco School of Medicine. Botulinum toxins (BoNT) are extremely potent neurotoxins produced by the anaerobic bacterium Clostridium botulinum. BoNT is a major biological threat agent that may be used by bioterrorists to threaten troops in the battlefield or civilians here at home. A polyvalent vaccine has been developed, but protective immunity takes months to develop and may be directed against only one or two of the seven distinct serotypes. Polyvalent equine or human immune globulin protect experimental animals and appear to protect humans against BoNT intoxication. However, equine immune globulin has a high incidence of side effects, including serum sickness and anaphylaxis, and human immune globulin is not available in large quantities. Dr. James Marks has generated murine and human single chain Fv (scFv) phage antibody libraries from individuals immunized against BoNT, and has identified three groups of scFv that bound non-overlapping epitopes on BoNT/A and neutralized toxin in vitro and in vivo. At present, plants offer the best system for the large scale, inexpensive production of these antibodies. Codon-optimized genes encoding the three best antibodies will be cloned into vectors designed for the expression of human IgG1 in transgenic plants. Plants will be transformed with these constructs, regenerated and screened for expression of antibody. Plants expressing high levels of the antibodies will be identified and used to purify enough to test for neutralization activity against BoNT. We estimate that plants will facilitate the production of metric ton quantities of antibodies at 1-5% of the cost of steel tank bioreactors. Plantibody technology will greatly reduce the costs to stockpile antibodies for protection of the population from this serious threat. These antibodies will also be available to treat the approximately 100 yearly cases of naturally occurring botulism in the USA.